The effect of microtubule disruption on the development and maintenance of cell polarity was studied in rat hepatocytes cultured as primary monolayers in the presence of colchicine or nocodazole. Addition of colchicine immediately after plating did not inhibit the generation of bile canaliculi (the apical pole) after 1 day of culture, as judged by electron microscopic examination, and did not allow penetration of Ruthenium Red through the tight junctions. However, the bile canaliculi developed in the presence of colchicine or nocodazole were not fully normal since they were not able to concentrate fluorescein diacetate in their lumina, and did not enrich with proteins of the apical plasma membrane domain, as control cells did. When the drugs were added after 1 or 2 days of culture, the new bile canaliculi appeared to be unaffected when examined by electron microscopy, but many of them did not concentrate fluorescein and were not enriched with apical membrane proteins within 4 to 24 h after drug addition. Whenever the drugs were added, the proteins that would normally concentrate on the membrane of the bile canaliculi accumulated intracellularly in endocytic vesicles after 2 to 4 h of drug treatment, and in vacuoles resembling lysosomes when the drugs were maintained for 24 h or more. These results show that microtubule disruption does not inhibit the structural reconstitution of bile canaliculi, but impairs their normal function and the transport of proteins of the apical plasma membrane domain.